The long-term goal of the parent grant is to determine the structural and functional determinants of paracellular permeation in epithelia. We previously developed a model using inducible overexpression of claudin-2 in MDCK I cells to study the properties of the claudin-2 cation pore. The parent grant had 3 specific aims, focused on characterizing the functional properties of claudin-2, identifying the basis of its cation selectivity, and mapping the structure of the pore. The impetus for this revision application is the recent discovery by our collaborator, Michael Fromm, that claudin-2 also substantially increases transepithelial water permeability. The current application seeks to expand the scope of the parent grant by adding a 4th specific aim, to characterize the mechanism of claudin-2-induced water permeability. This will generate new information that is itself entirely novel and physiologically significant, but will also add to the data generated by the other three specific aims to enhance our overall understanding of the structure of the pore. Furthermore, mutants generated in the original aims that alter ion selectivity and conductance can be exploited to probe the underlying mechanism of water permeability in this new aim. Thus, there will be substantial conceptual and experimental synergy between the proposed new aim and the original aims of the parent grant. This revision will accelerate the tempo of scientific research funded by the parent grant. It is expected to stimulate the economy by funding the hiring of a new graduate student, and procuring needed equipment for this project, including custom Ussing chambers for water flow measurements. PUBLIC HEALTH RELEVANCE: Principal Investigator: YU, Alan S. L. NARRATIVE This project seeks to understand the structure-function relationship of claudin-based pores and the mechanism for salt and water permeation. This may eventually lead to the identification of diseases that are due to abnormal paracellular permeability, which might include forms of salt-sensitive hypertension, salt-wasting, or diabetes insipidus. It may also lead to the development of drugs targeted to the claudin pore region that could modulate paracellular permeability and act as modulators of urinary electrolyte excretion.